CNS development requires a coordinated program that regulates the production and death of billions of neurons. Though the processes of cell division and cell death would seem to be polar opposite concepts, work during previous project periods has shown that in both development and disease, many instances of nerve cell death are accompanied by evidence for an abortive process of cell division. By both immunocytochemical and genetic analysis we have shown that the necessary proteins are made and the expected events of DNA replication are found in members of the neuronal populations that are at risk for death. This is true both in experimental systems and in two different human degenerative diseases: ataxia-telangiectasia and Alzheimer's disease. During the coming project period, these findings will be pursued. First, we will characterize our in vitro culture system to determine with high fidelity the nature of the cells that are present in vitro. We predict that only those cells that were actively dividing at the time of sacrifice survive the dissociation and plating process. This opens up the possibility of using age of harvest as a variable that permits assay of the behavior of individual strata of the neocortex in culture. Our second goal is to explore the Cdk5 kinase for its role in the process of cell death. Recent work implicates the Cdk5-dependent phosphorylation of the MEF2 transcription factor as a trigger for the transcription of pro-apoptotic genes. We will investigate this area using stimuli that cause neuronal death both with and without accompanying evidence of cell cycle processes. We will determine whether cell cycling and cell death can be dissociated by inhibition of Cdk5 activity, and whether the induction of Cdk5 activity and cell cycle initiation are independent or related processes. Finally, we will determine whether the induction of cell cycle enzymes and DNA replication that we observe in conjunction with cell death is reflective of a massive DNA repair process rather than true origin-based replication. The presence and location of replication complex proteins will be determined, and DNA combing techniques will be used to indicate the relationship of helix extension to known origins of replication. Transgenic mice will be produced that block the formation of the replication complex on the DNA. If origin based replication is required for cell death then the neurons should be resistant to cell cycle induced death.